JPH0226638B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel oligosaccharide having growth-promoting activity of Bifidobacterium and a method for producing the same.

Bifidobacterium (hereinafter referred to as Bifidobacterium) is a useful bacterium that grows in the human intestines, and it has been reported that it has various physiological and nutritional effects on humans. For example, inhibiting intestinal putrefaction, synthesizing vitamin _B1 and _B2 ,
Furthermore, effects on protein metabolism are known.

Therefore, it is important to promote the growth of Bifidobacterium in the human body in order to improve the above-mentioned effects.

Conventionally, many studies have been conducted for a long time on substances that promote the growth of Bifidobacterium (hereinafter referred to as Bifidobacterium growth factors), and the growth factors include N-acetylglucosamine, carrot extract,
Lactylose etc. have been reported. However, although these bifidus growth factors are effective in vitro, their in vivo effects are either unconfirmed or extremely insufficient results have been obtained.

In recent years, oligosaccharides have been attracting attention as bifidus growth factors, and the general formula is
It has been proposed to use an oligosaccharide represented by Gal-(Gal) _o -Glc (in the formula, Gal is a galactose residue, Glc is a glucose residue, and n is an integer from 1 to 4) as a bifidus growth factor. (Unexamined Japanese Patent Publication 1983)
−104885).

As a result of studying the effect of oligosaccharides as Bifidobacterium growth factors, the present inventor obtained the knowledge that the novel oligosaccharides shown below exhibit an excellent growth-promoting effect on Bifidobacterium in vivo. He came up with an invention.

Therefore, an object of the present invention is to provide a novel oligosaccharide as a bifidus growth factor and a method for producing the same. The present invention will be explained in detail below.

The oligosaccharide according to the present invention is a novel substance having the following formula ().

Oligosaccharides are produced by the galactosyl transfer reaction (transfer of galactoside bonds) that occurs when β-galactosidase acts on lactose. To date, the following oligosaccharides have been isolated and confirmed. There is.

β-Gal-(1→2)-Glc, β-Gal-(1→
3) -Glc, β-Gal- (1 → 6) -Glc, β-
Gal-(1→3)-Gal, β-Gal-(1→6)-
Gal, β-Gal-(1→6)-β-Gal-(1→
4) -Glc and β-Gal- (1 → 6) -β-Gal-
(1→6)-Glc (in the formula, Gal represents galactose and Glc represents glucose), etc. Also, as mentioned above, recently
An oligosaccharide represented by Gal-(Gal) _o -Glc (in the formula, n is an integer of 1 to 4) has been reported (Japanese Patent Application Laid-Open No. 1989-1999).
No. 104885).

The difference between these known oligosaccharides and the oligosaccharide according to the present invention having the above formula () is that the former has glucose or galactose linked in a linear chain, whereas the latter has the above formula (). As you can see, two molecules of galactose are attached to glucose in a branched manner. That is, the oligosaccharide according to the present invention can be said to be a novel oligosaccharide that is structurally different from the above-mentioned known oligosaccharides in that two molecules of galactose are bonded to the carbons at the 4th and 6th positions of glucose. Next, the structure of the oligosaccharide according to the present invention will be explained.

(a) Molecular weight Measurement using a mass spectrometer shows a molecular weight of 504,
From the results, it is estimated that this oligosaccharide is a trisaccharide consisting of three molecules of hexose.

(b) Constituent sugar The molar ratio of the sugar produced by hydrolyzing this oligosaccharide with 0.5N-HCl at 100°C for 4 hours is glucose:galactose = 1:2, so this oligosaccharide is 1:2. It was confirmed that it is a trisaccharide consisting of one molecule of glucose and two molecules of galactose.

(c) Binding mode of constituent sugars This oligosaccharide was partially hydrolyzed with 0.1N HCl at 100℃ for 20 minutes, and the resulting decomposition mixture was analyzed by thin layer chromatography, as shown in the attached Figure 1. showed a pattern. From this result, it was found that O-β-D-galactosyl-(1
→4) Two types of -D-glucose and O-β-D-galactosyl-(1→6)-D-glucose
Sugars were confirmed.

From the results of (a) to (c) above, it is clear that the oligosaccharide according to the present invention has the formula () It can be identified as nosyl-(1→6)]-D-glucose.

In addition, this oligosaccharide exhibits the following physical and chemical properties.

1 Solubility in solvents: Easily soluble in water, insoluble in acetone, alcohol, chloroform, and benzene, and slightly soluble in hydrous alcohol.

2 Color reaction: Aniline/phthalate reaction is positive, ninhydrin reaction is negative.

3 Distinction between basic, acidic, and neutral: neutral 4 Color tone: The dry powder is white in color.

Next, the method for producing oligosaccharides according to the present invention will be explained.

In the production method of the present invention, first, β-galactosidase is allowed to act on lactose or a lactose-containing substance to perform a galactose transfer reaction, thereby producing a reaction mixture consisting of a mixture of oligosaccharides. The lactose used as a starting material here may be commercially available, and may include whole milk, skim milk,
Lactose-containing substances such as whey may also be used as starting materials.

The origin of β-galactosidase to be applied to the above-mentioned starting material is not particularly limited, and it does not need to be highly purified, and can be used in the form of a crude enzyme.

In order to cause β-galactosidase to act on the above-mentioned lactose or lactose-containing substances, a starting material with a lactose concentration of 5 to 50% is used as a substrate, and a pH of 2 to 50% is used as a substrate.
8. It is appropriate to allow the enzyme to act at an enzyme concentration of 0.1 to 200 units/ml at a temperature of 10 to 60°C. In addition, since the reaction time for allowing the enzyme to act has a large effect on the yield of oligosaccharides, it is necessary to confirm the optimal reaction time by experiment. That is, the reaction time is adjusted while quantifying the oligosaccharide in the reaction mixture produced by the action of the enzyme using high performance liquid chromatography.

Through the above enzymatic reaction, a galactose transfer reaction occurs in the starting material to produce an oligosaccharide mixture.

In the present invention, when the enzyme reaction is completed, the enzyme is inactivated by heating the reaction solution at 90°C or higher for 2 to 30 seconds, and then the solution is passed through activated carbon to remove oligosaccharides or more of trisaccharides in the reaction solution. Adsorbs only sugar.

This adsorption treatment is for removing unreacted lactose, monosaccharides produced by decomposition, and various oligosaccharides produced by the transfer reaction from the reaction mixture to increase the concentration of the target oligosaccharide. Therefore, when flowing the activated carbon, it is necessary to control so that only oligosaccharides of trisaccharides or more in the reaction mixture are substantially adsorbed. For this purpose, the amount of the reaction mixture passed through the activated carbon column is controlled so that the amount of oligosaccharide containing trisaccharides or more in the reaction mixture is 50 to 100 g per 1 kg of activated carbon. Note that if the liquid is passed in an amount lower than the above range, the disaccharides in the reaction mixture will also be adsorbed by the activated carbon, whereas if the liquid is passed in an amount higher than this range, some of the target oligosaccharides will be eluted without being adsorbed. There is a need to.

The activated carbon used in the above-mentioned adsorption treatment may be one that is normally commercially available, or the activated carbon may be mixed with a supercharging agent such as celite. Furthermore, it is preferable to use the activated carbon in a state in which a suitable amount of water is added to form a slurry, which is filled into a column, and then a sufficient amount of water is passed through the column to equilibrate with water.

Next, 3 was adsorbed onto activated carbon as described above.
Elutes oligosaccharides greater than saccharides. Ethanol is usually used at a concentration of 15 to 50% for this elution, and the oligosaccharide of interest can thereby be efficiently eluted.

The eluate thus obtained contains the oligosaccharides targeted by the present invention, but it also contains other oligosaccharides of trisaccharides or more, so the present invention further includes the following steps. Add and refine.

That is, a solution obtained by concentrating the above eluate under reduced pressure, or a solution obtained by drying (for example, spray drying) and powdering the above solution and dissolving it in warm water is treated with β-galactosidase again to obtain oligosaccharides other than the desired oligosaccharide. Decomposes sugar. Next, the solution obtained by enzymatic decomposition is passed through the activated carbon column again so that only the desired oligosaccharide is adsorbed onto the activated carbon, and the adsorbed oligosaccharide is eluted in the same manner as above to obtain the purified product. Obtain the oligosaccharide of the invention.

The eluate thus obtained was concentrated under reduced pressure.
Further dry and powder if necessary.

The resulting powder is white and contains approximately 90% O-β-
D-galactopyranosyl-(1→4)-[O-β
-D-galactopyranosyl- (1→6)] -D-
Contains glucose.

The oligosaccharide obtained as described above according to the present invention exhibits an excellent growth-promoting effect on Bifidobacteria, as seen in the test results shown below.
The oligosaccharide according to the present invention can be used as a bifidus growth factor either in powder form or in the form of the above-mentioned concentrate.

Growth test for Bifidobacterium: Test method A group of 10 cynomolgus monkeys were used as the test animals, and each monkey was given commercially available powdered infant milk containing 5% by weight of lactose for 3 weeks. Powdered milk for infants containing 5% by weight of the monkeys was fed for an additional 3 weeks, during which time the feces of each monkey was collected and the proportion of Bifidobacterium in the feces was measured. In addition,
For comparison, an oligosaccharide mixture (powder form) of trisaccharides or more without the second stage of activated carbon adsorption treatment and subsequent elution treatment in the present invention was prepared in the same manner as above and tested in other groups of monkeys. The proportion of Bifidobacterium in the feces was measured. The results are shown in the attached Figure 2. As shown in Figure 2, compared to the amount of Bifidobacterium in the feces during the period when lactose was added, the amount of Bifidobacterium in feces during the period when the oligosaccharide mixture and the oligosaccharide of the present invention were added. The percentage of bacteria increased significantly, and the growth of Bifidobacterium was clearly improved in the samples fed with the oligosaccharide of the present invention compared to those fed with the oligosaccharide mixture.

That is, the oligosaccharide of the present invention has an extremely excellent effect on promoting the growth of Bifidobacterium in vivo, and also has an extremely excellent effect on promoting the growth of Bifidobacterium in the human intestinal tract, such as Bifidobacterium breve and Bifidobacterium. It exhibits high growth activity against a wide range of Bifidobacterium species such as Bacterium longum, Bifidobacterium bifidum, Bifidobacterium addressenteis, and Bifidobacterium infantis.

Therefore, the oligosaccharide according to the present invention can be incorporated into dairy products such as powdered milk and fermented milk, or added as a component of drugs such as intestinal preparations.

Examples of the present invention are shown below.

Example 45,000 units of β-galactosidase was added to a solution of 10 kg of lactose dissolved in 15 kg of warm water, the pH of which was adjusted to 4.5 by adding citric acid, and the mixture was reacted at 40°C for 2 hours. After heating the resulting reaction mixture at 105°C for 2 seconds to inactivate the enzyme,
40cm activated carbon column (25Kg of activated carbon and Celite)
12.5 kg of the mixture was made into a slurry with water) at a flow rate of 2 kg/hour. At this time, the amount of trisaccharide or higher oligosaccharides per 1 kg of activated carbon is 80 g. Next, a sufficient amount of water was passed through the solution to elute the monosaccharides and the two types in the reaction mixture, and then the oligosaccharides adsorbed on the activated carbon were eluted using 20% ethanol. The obtained eluate was concentrated under reduced pressure and then spray-dried to obtain a white powder (crude product). This powder contains O-β, which is the oligosaccharide of the present invention.
-D-galactopyranosyl-(1→4)-[O-
β-D-galactopyranosyl-(1→6)]-D
- Contains about 20% glucose by weight.

Then 100 g of the powder obtained as described above
was dissolved in 5 kg of warm water, 100 units of β-galactosidase was added to this solution, and the mixture was reacted at 40°C for 2 hours.

After deactivating the enzyme, the resulting reaction solution was heated to 10 cm.
×10cm activated carbon column (activated carbon and celite 2:
1 mixture) to adsorb oligosaccharides.
Next, this adsorbed oligosaccharide was eluted using 20% ethanol, and the resulting eluate was concentrated under reduced pressure and then lyophilized to obtain 20 g of white powder.

This powder contains approximately 90% by weight of O-β-D-galactopianocyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucose. Was.

Prepare powdered milk for infants to which 2% (weight) of the oligosaccharide powder of the present invention obtained as described above is added,
When this was administered to 30 infants within 3 months of birth, the ratio of bifidobacteria to the total number of bacteria in their feces increased approximately twice as much as when 2% (weight) of lactose was added. It was recognized that

[Brief explanation of drawings]

Figure 1 shows the thin layer chromatography pattern of the partial hydrolyzate of oligosaccharide according to the present invention, and Figure 2 is a graph showing the in vivo growth-promoting effect of the oligosaccharide according to the present invention. This is shown in .

Claims (1)

[Claims] 1. General formula O-β-D-galactopyranosyl-(1
→4) - [O-β-D-galactopyranosyl-
(1 → 6)] - Oligosaccharide consisting of D-glucose. 2. A step of causing β-galactosidase to act on lactose or a lactose-containing substance to perform a galactose transfer reaction to generate an oligosaccharide-containing reaction mixture, and passing the above reaction mixture through an activated carbon column to extract three types of oligosaccharides in the reaction mixture. A step of adsorbing the above oligosaccharide mixture on activated carbon, and then eluting the adsorbed oligosaccharide mixture, further adding β to the eluted oligosaccharide mixture.
- A formula characterized by a step of causing a decomposition reaction by causing galactosidase to act, passing the resulting reaction product through an activated carbon column to adsorb the target oligosaccharide, and then eluting the adsorbed oligosaccharide, O-β-D-galactopyranosyl-(1
→4) - [O-β-D-galactopyranosyl-
(1→6)] - A method for producing an oligosaccharide consisting of D-glucose. 3. Transfer the oligosaccharide-containing reaction mixture to an activated carbon column equilibrated with water.
The manufacturing method according to claim 2, wherein the liquid is passed through at a rate of 100 g. 4 formula O-β-D-galactopyranosyl-(1
→4) - [O-β-D-galactopyranosyl-
(1→6)] A growth promoter for Bifidobacterium containing an oligosaccharide consisting of -D-glucose as an active ingredient.